15 research outputs found
Building ProteomeTools based on a complete synthetic human proteome.
We describe ProteomeTools, a project building molecular and digital tools from the human proteome to facilitate biomedical research. Here we report the generation and multimodal liquid chromatography-tandem mass spectrometry analysis of \u3e330,000 synthetic tryptic peptides representing essentially all canonical human gene products, and we exemplify the utility of these data in several applications. The resource (available at http://www.proteometools.org) will be extended to \u3e1 million peptides, and all data will be shared with the community via ProteomicsDB and ProteomeXchange
The morphology and biochemistry of nanostructures provide evidence for synthesis and signaling functions in human cerebrospinal fluid
<p>Abstract</p> <p>Background</p> <p>Cerebrospinal fluid (CSF) contacts many brain regions and may mediate humoral signaling distinct from synaptic neurotransmission. However, synthesis and transport mechanisms for such signaling are not defined. The purpose of this study was to investigate whether human CSF contains discrete structures that may enable the regulation of humoral transmission.</p> <p>Methods</p> <p>Lumbar CSF was collected prospectively from 17 participants: with no neurological or psychiatric disease, with Alzheimer's disease, multiple sclerosis, or migraine; and ventricular CSF from two cognitively healthy participants with long-standing shunts for congenital hydrocephalus. Cell-free CSF was subjected to ultracentrifugation to yield supernatants and pellets that were examined by transmission electron microscopy, shotgun protein sequencing, electrophoresis, western blotting, lipid analysis, enzymatic activity assay, and immuno-electron microscopy.</p> <p>Results</p> <p>Over 3,600 CSF proteins were identified from repeated shotgun sequencing of cell-free CSF from two individuals with Alzheimer's disease: 25% of these proteins are normally present in membranes. Abundant nanometer-scaled structures were observed in ultracentrifuged pellets of CSF from all 16 participants examined. The most common structures included synaptic vesicle and exosome components in 30-200 nm spheres and irregular blobs. Much less abundant nanostructures were present that derived from cellular debris. Nanostructure fractions had a unique composition compared to CSF supernatant, richer in omega-3 and phosphoinositide lipids, active prostanoid enzymes, and fibronectin.</p> <p>Conclusion</p> <p>Unique morphology and biochemistry features of abundant and discrete membrane-bound CSF nanostructures are described. Prostaglandin H synthase activity, essential for prostanoid production and previously unknown in CSF, is localized to nanospheres. Considering CSF bulk flow and its circulatory dynamics, we propose that these nanostructures provide signaling mechanisms <it>via </it>volume transmission within the nervous system that are for slower, more diffuse, and of longer duration than synaptic transmission.</p
Triple Quadruple Mass Spectromery-Based Peptide Assays Using Intelligent SRM (iSRM)
Comunicaciones a congreso
PPKs mediate direct signal transfer from phytochrome photoreceptors to transcription factor PIF3.
Upon light-induced nuclear translocation, phytochrome (phy) sensory photoreceptors interact with, and induce rapid phosphorylation and consequent ubiquitin-mediated degradation of, transcription factors, called PIFs, thereby regulating target gene expression and plant development. Nevertheless, the biochemical mechanism of phy-induced PIF phosphorylation has remained ill-defined. Here we identify a family of nuclear protein kinases, designated Photoregulatory Protein Kinases (PPK1-4; formerly called MUT9-Like Kinases (MLKs)), that interact with PIF3 and phyB in a light-induced manner in vivo. Genetic analyses demonstrate that the PPKs are collectively necessary for the normal light-induced phosphorylation and degradation of PIF3. PPK1 directly phosphorylates PIF3 in vitro, with a phosphosite pattern that strongly mimics the light-induced pattern in vivo. These data establish that the PPKs are directly involved in catalysing the photoactivated-phy-induced phosphorylation of PIF3 in vivo, and thereby are critical components of a transcriptionally centred signalling hub that pleiotropically regulates plant growth and development in response to multiple signalling pathways
Sensitive and Accurate Quantitation of Phosphopeptides Using TMT Isobaric Labeling Technique
Phosphorylation
is an essential post-translational modification
for regulating protein function and cellular signal transduction.
Mass spectrometry (MS) combined with isobaric tandem mass tags (TMTs)
has become a powerful platform for simultaneous, large-scale phospho-proteome
site identification and quantitation. To improve the accuracy of isobaric
tag-based quantitation in complex proteomic samples, MS3-based acquisition
methods such as Synchronous Precursor Selection (SPS) have been used.
However, the method suffers from lower peptide identification rates
when applied to enriched phosphopeptide samples compared with unmodified
samples due to differences in phosphopeptide fragmentation patterns
during tandem MS. We developed and optimized two new acquisition methods
for analysis of TMT-labeled multiplexed phosphoproteome samples, which
resulted in more phosphopeptide identifications with less ratio distortion
when compared with previous methods. We also applied these improved
methods to a large-scale study of phosphorylation levels in A549 cell
lines treated with insulin or insulin growth factor 1 (IGF-1). Overall,
3378 protein groups and 12 465 phosphopeptides were identified,
of which 10 436 were quantified across 10 samples without prefractionation.
The accurate measurement enabled us to map to numerous signaling pathways
including mechanistic target of rapamycin (mTOR), epidermal growth
factor receptor (EGFR, ErbB), and insulin signaling pathways
Intact NIST Monoclonal Antibody Characterization - Proteoforms, Glycoforms - using CE-MS and CE-LIF
10.1080/23312009.2018.1480455Cogent Chemistry41148045
Trimodal Mixed Mode Chromatography That Enables Efficient Offline Two-Dimensional Peptide Fractionation for Proteome Analysis
Offline two-dimensional chromatography
is a common means to achieve
deep proteome coverage. To reduce sample complexity and dynamic range
and to utilize mass spectrometer (MS) time efficiently, high chromatographic
resolution of and good orthogonality between the two dimensions are
needed. Ion exchange and high pH reversed phase chromatography are
often used for this purpose. However, the former requires desalting
to be MS-compatible, and the latter requires fraction pooling to create
orthogonality. Here, we report an alternative first-dimension separation
technique using a commercial trimodal phase incorporating polar embedded
reversed phase, weak anion exchange, and strong cation exchange material.
The column is capable of retaining polar and nonpolar peptides alike
without noticeable breakthrough. It allows separating ordinary and
TMT-labeled peptides under mild acidic conditions using an acetonitrile
gradient. The direct MS compatibility of solvents and good orthogonality
to online coupled C18 columns enable a straightforward workflow without
fraction pooling and desalting while showing comparable performance
to the other techniques. The method scales from low to high microgram
sample quantity and is amenable to full automation. To demonstrate
practical utility, we analyzed the proteomes of 10 human pancreatic
cancer cell lines to a depth of >8,700 quantified proteins
Intact NIST monoclonal antibody characterization—Proteoforms, glycoforms—Using CE-MS and CE-LIF
<p>Determining and linking the structural heterogeneity of recombinant antibodies to function is critical in the biopharmaceutical industry. We introduce a new microfluidic capillary electrophoresis—mass spectrometry (μCE-MS) approach to characterize intact monoclonal antibody (mAb) and simultaneously quantifying distinct variants. Our MS analysis of intact NIST mAb (RM8671) shows 18 variants identified amongst proteolytic and glycolytic modifications with a range of relative abundances between 0.1% and 100%. In order to verify our quantitative MS results, we used an established system based on capillary electrophoresis—with laser induced fluorescence (CE-LIF) for profiling the N-glycans. All major glycans were identified and further substantiated by exoglycosidase digestion. Interestingly, the µCE-MS analysis of intact NIST mAb consistently yielded higher amounts of G2FG2F-Hex glycoform (~3.4%), whereas the CE-LIF analysis indicates that only 1.4% of G2F-Gal is present. Therefore, the additional hexose adduct observed in µCE-MS may have been the glycation product of the mAb. Further analysis of deglycosylated mAb with µCE-MS made it possible to reveal the glycation with 10.5% of one hexose product and 4.9% of two hexose product in the intact deglycosylated antibody. An integrated solution using two orthogonal and complementary techniques for characterizing antibody glycosylation is provided here.</p
High Performance Anion Exchange and Hydrophilic Interaction Liquid Chromatography Approaches for Comprehensive Mass Spectrometry-Based Characterization of the N‑Glycome of a Recombinant Human Erythropoietin
Comprehensive characterization
of the N-glycome of a therapeutic
is challenging because glycans may harbor numerous modifications (e.g.,
phosphorylation, sulfation, sialic acids with possible O-acetylation).
The current report presents a comparison of two chromatographic platforms
for the comprehensive characterization of a recombinant human erythropoietin
(rhEPO) N-glycome. The two platforms include a common workflow based
on 2-AB-derivatization and hydrophilic interaction chromatography
(HILIC) and a native N-linked glycan workflow employing high performance
anion exchange (HPAE) chromatography. Both platforms were coupled
to an Orbitrap mass spectrometer, and data dependent HCD fragmentation
allowed confident structural elucidation of the glycans. Each platform
identified glycans not revealed by the other, and both exhibited strengths
and weaknesses. The reductive amination based HILIC workflow provided
better throughput and sensitivity, had good isomer resolution, and
revealed the presence of O-acetylated sialic acids. However, it exhibited
poor performance toward phosphorylated glycans and did not reveal
the presence of sulfated glycans. Furthermore, reductive amination
introduced dehydration artifacts and modified the glycosylation profile
in the rhEPO glycome. Conversely, HPAE provided unbiased charge classification
(sialylation levels), improved isomer resolution, and revealed multiple
phosphorylated and sulfated structures, but delivered lower throughput,
had artifact peaks due to epimer formation, and loss of sialic acid
O-acetylation. The MS<sup>2</sup> based identification of phosphorylated
and sulfated glycans was not possible in HILIC mode due to their
poor solubility caused by the high acetonitrile concentrations employed
at the beginning of the gradient. After analyzing the glycome by both
approaches and determining the glycans present, a glycan library was
created for site specific glycopeptide analyses. Glycopeptide analyses
confirmed all the compositions annotated by the combined use of 2-AB-
and native glycan workflows and provided site specific location of
the glycans. These two platforms were complementary and in combination
delivered a more thorough and comprehensive characterization of the
rhEPO N-glycome, supporting regulatory conformance for the pharmaceutical
industry